Selective specificity of calcium-binding proteins calbindin and calretinin expression in the magnocellular neurosecretory hypothalamic nuclei of tortoises and turtles.

We have studied the distribution of calcium-binding proteins in the magnocellular neurosecretory nuclei of nonapeptidergic neurosecretory nuclei of the preoptic-hypothalamic complex in a tortoise (Testudo horsfieldi) and a pond turtle (Emys orbicularis) using immunohistochemistry. We have found that different types of cells in the paraventricular and supraoptic nuclei predominantly express calbindin and, to a lesser extent, calretinin, but not parvalbumin. The selective calbindin/calretinin control of the neurohormone secretion in these hypothalamic nuclei is an evolutionary conservative feature typical of reptiles and mammals.

Distribution of calcium-binding proteins, parvalbumin and calbindin, in the midbrain auditory center (MLd) of a pigeon.

Immunohistochemical distribution of calcium-binding proteins, parvalbumin (PV) and calbindin (CB), has been studied in the mesencephalic auditory center (MLd) of pigeon (Columba livia). In the central region of the MLd (core, ICC), an overlap in distribution of the PVand CB-immunopositive (ip) neurons and neuropil has been observed, with different patterns in the central and peripheral parts. In the peripheral region of the MLd (belt, ICS, and ICX), both neurons and neuropil contained only CB. A selective CB chemospecificity of the belt, ICS, and ICX is an evolutionary conserved feature characteristic of all avian species. Interspecies differences in the distribution of PV and CB immunoreactivity in the ICC are a result of adaptive functional specialization, which provides specific processing of different aspects of the auditory information.

[NEURONS IN VISUAL THALAMIC CENTERS OF TURTLES, PROJECTING UPON THE TELENCEPHALON, EXPRESS DIFFERENT TYPES OF CALCIUM-BINDING PROTEINS: COMBINED IMMUNOHISTOCHEMICAL AND TRACER STUDY].

In turtles (Testudo horsfieldi, Emys orbicularis) the immunoreactivity for calbindin (CB), parvalbumin (PV), calretinin (CR), and colocalization of PV and CB in neurons of visual thalamic nuclei (Rot, GLD) projecting to the telencephalon were studied using combined immunohistochemical and tracer method. The predominance of CB-ir neurons in Rot, CB-ir and CR-ir neurons in GLD and a lower amount of PV-ir neurons in both nuclei were shown. With double labeling fluoroimmunohistochemistry technique the colocalization of PV with CB was revealed in the majority of PV-ir neurons and in a fewer number of CB-ir neurons within both nuclei. After delivery of horseradish peroxidase into the telencephalic projection fields of Rot and GLD, retrograde labeling was found in the corresponding thalamic projection neurons immunoreactive for every protein investigated. After fluorescent tracer (Fluoro Gold) injection into the same telencephalic regions retrograde labeling was observed in Rot and GLD neurons, immunoreactive only for PV or CB, as well in neurons with colocalization of the both proteins. These data support predominance of CB-ir component in the rotundo-telencephalic pathway and CB/CR components in the geniculo-telencephalic pathway in turtles. The role of functional specialization in segregation of neurons expressing different types of calcium-binding proteins is postulated.

[Turtle isthmic complex of visual nuclei: immunohistochemical study of gamma-aminobutyric acid, choline acetyltransferase, calcium-binding proteins and cytochrome oxidase activity].

The distribution of the immunoreactivity for gamma-aminobutyric acid (GABA), choline acetyltransferase (ChAT), calcium-binding proteins (CaBPr) and histochemistry of cytochrome oxidase activity (CO) was studied in turtles (Testudo horsfieldi, Emys orbicularis) isthmal complex of visual nuclei. Magnocellular nucleus (IMc) was shown to reveal mainly the strongly stained GABA-, parvalbumin (PV)-ir neurons and CO-positive cells, as well as variable both in number and degree of intensity of ChAT-, cal- bindin (CB)-, and calretinin (CR)-ir cells. After the local tracer injection into the optic tectum GABA-ir neurons containing also retrograde label were found in IMc. The most caracteristic of the parvocellular nucleus (IPc) was the content of strongly stained ChAT-ir neurons, dense GABA-ir and CO-active terminal fields, as well as the neurons variable by the amount and the degree of immunoreactivity for CaBPr and GABA. Principal similarity in these features in the turtle IMc and IPc and of those in the avian isthmal nuclei of the same name allows suggesting their homology and consequently the same participation in selective processing of the visual information flow. The comparison with lower vertebrates confirms the evolutionary conservatism of visual isthmal complex among vertebrates and the existence of its progressive differentiation in the process of evolution.

[Calcium-binding proteins and cytochrome oxidase activity in the turtle optic tectum characterzing the visual tectofugal pathway].

Using immunohistochemistry and tracer technique, we studied in the optic tectum of turtles (Emys orbicularis and Testudo horsfieldi) the distribution of CaBPr (parvalbumin, PV, calbindin, CB, calretinin, CR) before and after horseradish peroxidase delivery into nucleus rotundus (Rot). In parallel, activity of cytochrome oxidase (CO) was studied. In the main link of the tectofugal visual pathway (the central gray layer, SGC) in the both chelonian species rare PV-ir, as well CB- and CR-ir neurons were found to vary significantly both in the number and the immunoreactivity degree of their bodies and dendrites. The superficial (SGFS) and deep periventricular (SGP) tectal layers, on the contrary, contained numerous cells immunoreactive to all three CaBPr in different quantitative proportions. Only a small part of the retrogradely labeled tectorotundal neurons contained PV, CB or CR. Very large PV-ir neurons were not retrogradely labeled; by their morphological characteristics, they corresponded to efferent neurons with descending projections. SFC neurons of two chelonian species differed in the degree of CO activity. In SGFS, dense immunoreactivity of neuropil to all three CaBPr and the high CO activity were observed in both species with some differences in sublaminar distribution for every of proteins. Peculiarities of distribution of CaBPr-ir and of CO activity in various segments of SGC neurons are discussed with respect to laminar organization of the turtle tectum and to patterns of its retinal innervations. It was proposed that in projectional tectorotundal SGC neurons, the studied CaBPr are concentrated mainly in their distal dendrites contacting with retinal afferents in the superficial retinorecipient tectal layer.

[Neurochemical characteristics of the turtle optic tectum: comparison with other reptilian species and birds].

Data on distribution of biologically active substances in the turtle optic tectum are compared with results of similar experiments on other reptilian as well as on avian species. In two turtle species (Testudo horsfield and Emys orbicularis), immunoreactivity to monoamines (5-HT and TH), NPY, as well as NADPH-d activity were similarly distributed in neuropil of the SGFS retinorecipient part and in that of the SGP/SAP periventricular layers. Immunoreactivity to neuropeptides SP and m-Enk was maximal in neuropil of the SGFS non-retinorecipient part. The periventricular layers were characterized by the abundant radial SP- and mENK-ir as well as the NADPH-d-positive neurons. Diffusely dispersed ChAT-ir elements and many ir fibers perpenducilar to the tectal surface were observed in the SGFS retinorecipient part; the SGFS non-retinorecipient part contained a dense plexus of thick ir fibers and diffusely distributed ir terminals. The GABA ir cells were the most numerous in the tectum; they were spread in all tectal layers. Thus, various biologically active substance located in superficial retinorecipient tectal sublayers could affect processing and transmission of information via ascending dendrites of neurons in deeper layers. The cells containing SP, m-Enk, and NADPH-d had laminar organization in SGP; via the system of ascending and descending axons, they are able to affect other structures within and outside of the optic tectum. Putative sources of tectal modulatory innervation are discussed. In all studied reptilian and avian species, the principal similarity is revealed in the neurochemical organization. Some differences might be explained by the level of tectal differentiation due to factors of phylogenetic evolution and/or adaptive specialization.

[Calcium-binding proteins and metabolic activity in thalamotelencephalic parts of the turtle visual system].

Distribution of three calcium-binding proteins (CaBPr) calbindin (CB), calretinin (CR) and parvalbumin (PV) in parallel with metabolic activity (cytochrome oxidase, CO) was studied in telencephalic projection zones of the tecto- and thalamofugal visual pathways in experiments on the Horsfield's terrapin Testudo horsfieldi and the pond turtle Emys orbicularis. It was shown that the nucleus rotundus (Rot) and dorsal lateral geniculate nucleus (GLd) terminal fields in both zones (dorsolateral region of the anterior ventricular ridge, Advrdl and dorsolateral cortex, Cxdl, respectively) were CB-immunoreactive (-ir) in the both studied turtle species. The highest density of CB-ir terminals and the focus of rotundal projections in the Advrdl core coincided precisely. The GLd terminal field in Cxdl also was CR-ir. The PV contribution to innervation of both projectional zones was much lower, especially to innervation of Cxdl from GLd. In spite of similar CB-ir innervation, the projectional field of the tectofugal pathway of Advrdl had the much higher CO activity than of that of the thalamofugal pathway in Cxdl. The neurons immunoreactive to all three CaBPr types were distributed in Cxdl in different ratios in each of layers. In the visual Advrdl area the overwhelming majority were PV-ir neurons, whereas CB-ir neurons were absent. The conclusion is made that in spite of the CB- or CB/CR-immunoreactivity predominates over the PV-immunoreactivity in both thalamotelencephalic pathways of the visual system, the tectofugal (rotundo-Advrdl) pathway having the higher metabolic activity.

[Components of the pigeon tectothalamic visual pathway, revealed with aid of study of cytochrome oxidase and immunoreactivity to calcium-binding proteins].

Distribution of activity of cytochrome oxidase (CO) and immunoreactivity to parvalbumin (Pv) and calbindin (Cb) was studied in the optic tectum of the pigeon (Columba livia). In the first link of the tectofugal pathway in the central gray layer (SGC = layer 13), small amounts of the CO-active and Pv-immunoreactive (Pv-ir) cellular bodies were revealed in its internal part (sublayer 13b). Some of these neurons located along the SGC lower boundary had long dendritic processes ascending into the superficial tectum layer (SGF). In the retinorecipient SGF sublayers and particularly in neuropil of the sublayers 4 and 7, the high CO activity correlating with Pv-immunoreactivity was found. It is suggested that a great contribution to metabolic activity of these sublayers is made by the largely branching dendritic processes of Pv-ir neurons of sublayer 13b. The projectional neurons SGC located in its external part (sublayer 13a) were CO-inactive and contained Cb. They sent long dendrites into sublayer 5b; in its neuropil, the high density of Cb-immunoreactivity and a moderate CO activity were detected. Thus, the tectal link of the pigeon tectofugal visual channel consists of two components--the Pv-specific, highly metabolically active and the Cb-specific, metabolically less active ones that transduce visual information from different retinorecipient SGF sublayers. The absence of the significant amount of CO-positive bodies of projectional neurons in SGC can be due to that metabolically more active are their dendritic arborizations in the SGF sublayers.

[Evolutionary significance of reciprocal connections in the turtle tectofugal visual system].

In two turtle species--Emys orbicularis and Testudo horsfieldi--by the method of anterograde and retrograde traicing method at the light and electron microscopy level, the existence is proven of direct descending projections from the thalamic nucleus of the tectofugal visual system n. rotunds (Rot) to the optic tectum. After injection of tracers into Rot alone and into Rot with involvement of the tectothalamic tract (Trtth), occasional labeled fibers with varicosities and terminals are revealed predominantly in the deep sublayers of SGFS of the rostral optic tectum, while in the lower amount in other tectal layers. After the tracer injections into the optic tectum, a few retrogradely labeled neurons were found mainly in the Rot ventral parts and within Trtth. Their localization coincides with that of GABA-immunoreactive cells. Electron microscopy showed the existence of many retrogradely labeled dendrites throughout the whole Rot; a few labeled cell bodies were also present there, some of them being also GABA-immunoreactive. These results allow us to conclude about the existence of reciprocal connections between the optic tectum and Rot in turtles, these connections being able to affect processing of visual information in tectum. We suggest that reciprocity of tectothalamic connections might be the ancestral feature of the vertebrate brain; in the course of amniote evolution the functional significance of this feature can be decreased and even lost in parallel with a rise of the role of direct corticotectal projections.

[Metabolic activity of thalamic and telencephalic auditory centers in the pigeon].

Distribution of activity of the mitochondrial oxidative enzyme cytochrome oxidase C was studied in the thalamic (Ov) and telencephalic (field L) auditory centers in pigeons. The CO activity level has been shown to differ in the central (core) and peripheral (belt) subdivisions of these centers: the high CO activity in the former (nCe, L2) and the much lower or absent in the latter (Ovl, Ovm, SPO). Comparison of our data with those of various avian and reptile species confirms the concept of the common plan of rostral auditory centers in sauropsid amniotes by the principle of the center-periphery (core-belt), which is characteristic of the corresponding mammalian centers. The separation of the central and peripheral parts of these centers is better pronounced in birds than in reptiles.

[Distribution of metabolic activity (cytochrome oxidase) and immunoreactivity to calcium-binding proteins in turtle brainstem auditory nuclei].

Using histochemical and immunohistochemical techniques, distribution of activity of oxidative mitochondrial enzyme cytochrome oxidase (CO) and of immunoreactivity to calcium-binding proteins has been studied in spiral ganglion and auditory nuclei of brainstem in two turtle species. It has been shown that immunoreactivity to calbindin, parvalbumin, and calretinin in neurons and neuropil of nuclei of cochlear and superior olivary complexes, in nucleus of lateral lemniscus, and in spiral ganglion neurons coincides topographically with the high CO activity. The similarity of the studied metabolic and neurochemical characteristics of these auditory centers in reptiles, birds, and mammals indicates the existence of some common principles of their organization in amniotes in spite of phylogenetic differences and peculiarities of specialization of the auditory system in different species.

[The level of metabolic activity (cytochrome oxidase) as an index of functional significance of tectofugal and thalamofugal channels of the reptilian visual system].

Using histochemical determination of activity of the mitochondrial oxidative enzyme cytochrome oxidase (CO) in brain structures, metabolic activity both in turtles and in lizards has been shown to be higher in centers of the tectofugal channel (the tectal stratum griseum centrale, SGC; nucleus pretectalis ventralis, Ptv; thalamic nucleus rotundus, Rot; telencephalic visual area of the anterior dorsal ventricular ridge, Advr) than in the thalamofugal channel centers (the thalamic nucleus geniculatus lateralis pars dorsalis, GLd; cortex dorsolateralis, Cxdl; and pallial thickening, Path) of the visual system. Some interspecies differences in distribution of the CO activity in the tectal, thalamic, and telencephalic visual centers between terrestrial and pond turtles and lizards were revealed. The obtained data confirm the idea on the dominating role of the tectofugal channel over the thalamofugal channel of the visual system in information processing and organization of the day-to-day behavior of reptiles.

[Extrinsic cells, immunoreactive to Ca-binding protein, as sources of thalamic visual centres in tortoises].

Extrinsic sources of calcium-binding proteins involved in immunoreactive innervation of the visual thalamic nuclei Rot and GLd in turtles (Testudo horsfieldi and Emys orbicularis) were studied using HRP tracing method and immunohistochemistry. In 1.5-4.5 months after monocular enucleation calbindin (Calb)-, parvalbumin (Parv)- and calretinin (Calr)-labeling was found in fragments of degenerated retinal fibers in the contralateral optic tract and in some retinorecipient structures (optic tectum, GLd and GLv). Changes in GLd were detected in its neuropil part. in 2.0-3.5 months after unilateral ablation of tectum and pretectum, the densities of Parv-, Calb- and Aclr-immunoreactivity terminals and fibers were diminisched in the ipsilateral n. Rot, with the maximum effect seen in Parv. Following HRP injection into the visual thalamus (Rot and GLd), retrogradely labeled cells with Parv label only, were revealed in the ventrothalamic nucleus Enta, pretectal nucleus Ptv, and in all types of Ca-binding proteins (CaBPr) in separately labeled cells of the optic tectum. Thus, it has been shown that thalamic visual centers in turtles have multiple extrinsic cells, which serve as sources of CaBPr projections. The present data suggest that organization of CaBPr inputs to visual thalamus in reptiles (turtle) and higher amniotes are fundamentally similar.

Pretectal connections in turtles with special reference to the visual thalamic centers: a hodological and gamma-aminobutyric acid-immunohistochemical study.

Projections of the pretectal region to forebrain and midbrain structures were examined in two species of turtles (Testudo horsfieldi and Emys orbicularis) by axonal tracing and immunocytochemical methods. Two ascending gamma-aminobutyric acid (GABA)ergic pathways to thalamic visual centers were revealed: a weak projection from the retinorecipient nucleus lentiformis mesencephali to the ipsilateral nucleus geniculatus lateralis pars dorsalis and a considerably stronger projection from the nonretinorecipient nucleus pretectalis ventralis to the nucleus rotundus. The latter is primarily ipsilateral, with a weak contralateral component. The interstitial nucleus of the tectothalamic tract is also involved in reciprocal projections of the pretectum and nucleus rotundus. In addition, the pretectal nuclei project reciprocally to the optic tectum and possibly to the telencephalic isocortical homologues. Comparison of these findings with previous work on other species reveals striking similarities between the pretectorotundal pathway in turtles and birds and in the pretectogeniculate pathway in turtles, birds, and mammals.

Retinal and cortical afferents to the dorsal lateral geniculate nucleus of the turtle, Emys orbicularis: a combined axonal tracing, glutamate, and GABA immunocytochemical electron microscopic study.

The dorsal lateral geniculate nucleus (GLd) of the turtle Emys orbicularis has been analyzed with axonal tracing methods and immunocytochemical techniques for glutamate (GLU) and gamma-aminobutyric acid (GABA), in combination with a quantitative study of the morphologic characteristics, distribution, and synaptology of the retinofugal and corticofugal terminals. Ultrastructural observations show that the vast majority of retinal terminals (Rtr) have clear, rounded synaptic vesicles and account for 16% of all profiles containing synaptic vesicles (PCSV). Their synaptic index (0.5) is low, and they make three times more contacts with the dendrites of projection cells than with those of interneurons. A low proportion of retinal terminals of a second category contain pleomorphic synaptic vesicles and are highly GABA immunoreactive. Axon terminals, unlabeled after intraocular injection of the tracer (SR), smaller in size and with more rounded clear synaptic vesicles, longer synaptic differentiations, and higher synaptic index than Rtr terminals, account for 19.7% of all PCSV and make asymmetric synaptic contacts with large dendrites of projection cells and less with the dendrites of interneurons. Some SR have been unambiguously identified as corticofugal terminals (Cg), either after cortical injection of the tracer (16%) or cortical lesion (37%). Retinal and Cg/SR terminals are spatially segregated within the GLd. Both are highly GLU immunoreactive, with the highest density of labeling over synaptic vesicles, suggesting that these terminals may use GLU as neurotransmitter. The level of GLU immunoreactivity of GABA-positive profiles is half that of Rtr and Cg/SR terminals and is greatest over mitochondria, possibly reflecting the 'metabolic' pool of GLU that serves as a precursor in the formation of GABA.

Fine structure of the dorsal lateral geniculate nucleus of the turtle, Emys orbicularis: a Golgi, combined HRP tracing and GABA immunocytochemical study.

The afferent and efferent cortical projections of the dorsal lateral geniculate nucleus (GLD) of adult specimens of the turtle Emys orbicularis were investigated after intraocular or intracortical injections of horseradish peroxidase (HRP), and the distribution of gamma aminobutyric acid (GABA) immunoreactivity in the nucleus was carried out by immunocytochemical techniques, both techniques being combined with light and electron microscopy. In addition, some specimens were prepared for double-labeling of HRP and GABA immunoreactivity, and additional samples impregnated by a rapid Golgi technique. On purely morphological grounds, four types of neurons can be distinguished by light microscopy: two types of large cells in the cell plate which project to the cortex, and two types of smaller cells in the neuropil and optic tract which do not. The small cells are consistently GABA-immunoreactive, while the former are, with extremely rare exceptions, immunonegative for GABA. The supposition that the small neurons of the neuropil are interneurons is supported by electron microscopic observations; these strongly GABA-immunoreactive cells have large plicated nuclei surrounded by a thin layer of cytoplasm poorly endowed with organelles. The dendrites of these cells may contain pleomorphic synaptic vesicles (DCSVs) and appear to be presynaptic to other dendritic profiles. These DCSVs are occasionally contacted by GABA-immunoreactive axon terminals, and more frequently by retinal terminals consistently immunonegative for GABA. The latter, frequently organized in glomeruli, also make synaptic contacts with immunonegative dendrites arising from corticopetal neurons of the cell plate. Two major categories of GABA-immunoreactive axon terminals can be distinguished, and we are led to the conclusion that one of these represents an intrinsic GABAergic innervation of the GLD, while the second is tentatively interpreted as an extrinsic source of GABA to the nucleus, possibly from ventral thalamic structures. The fine structure of the dorsal lateral geniculate nucleus of Emys orbicularis thus shows many similarities with that of mammals.

Evolutionary significance of different neurochemical organisation of the internal and external regions of auditory centres in the reptilian brain: an immunocytochemical and reduced NADPH-diaphorase histochemical study in turtles.

An immunocytochemical and histochemical study was undertaken of the torus semicircularis and nucleus reuniens, the mesencephalic and diencephalic auditory centres, in two chelonian species, Testudo horsfieldi and Emys orbicularis. The nucleus centralis of the torus semicircularis receives few 5-HT-, TH-, substance P-, and menkephalin-immunoreactive fibres and terminals, in marked contrast to the external nucleus laminaris of the torus semicircularis, in which 5-HT-, TH-, substance P-, and menkephalin-immunoreactive elements and cell bodies show a laminar distribution. Dense NPY-positive terminal-like profiles and cell bodies were observed in both the nuclei centralis and laminaris, and many NADPH-d-positive cell bodies were observed in the cell layers of the latter. In the nucleus reuniens, the distribution of 5-HT-, TH-, substance P-, and menkephalin-immunolabelling resembles that seen in the torus semicircularis, but at a lower density. The dorsorostral regions of the nucleus reuniens, as in the nucleus centralis, is insignificantly labelled, in contrast to the ventrocaudal regions in which labelled elements abound. NPY-positive elements are uniformly distributed throughout the nucleus, but no labelled cell bodies were observed. NADPH-d-positive fibres and terminals were observed in both dorsal and ventral regions of the nucleus reuniens, but the few labelled cell bodies to be observed were located in the peripheral regions of the nucleus. These findings are discussed in terms of the evolution of the core-and-belt organisation of sensory nuclei observed in other vertebrate species.

An underestimated visual pathway in reptiles.

Field potentials evoked in the turtle general cortex by electric stimulation of the optic tectum were analyzed. Cathodal polarization and subtotal lesions of n. rotundus led to either facilitation or depression of later components of the cortical potential. On the contrary, cathodal polarization and lesions of n.geniculatus lateralis dorsalis similarly affected the initial component. After 21-23 months following eye enucleation or optic nerve section, the conduction of fast tectal volleys to the general cortex via n. geniculatus lateralis dorsalis was blocked, though the conduction of tectal impulses to the dorsal ventricular ridge via n.rotundus survived. It is concluded that in turtles one more visual channel, i.e., retino-tecto-geniculo-cortical, is functioning, in addition to well-known retino-tecto-rotundo-telencephalic and retino-geniculo-cortical channels.

[Tecto-thalamo-telencephalic interrelationships following visual deafferentation]. / Tekto-talamo-teléntsefal'nye vzaimootnosheniia u cherepakh posle zritel'noi deafferentatsii.

21-23 months after uni- and bilateral enucleation of the eyes, the turtles Emys orbicularis and Testudo horsfieldi exhibited atrophy, gliosis and residual degenerative changes in the visual nerves and tracts and in the two upper layers of the optic tectum, as well as a reduced density of fibres in the tecto-thalamic tract on the deafferentated side. Electrophysiological experiments on the turtles have shown that tectal impulses en route to the general cortex of the forebrain are relayed in the n. rotundus. Conduction of impulses along the tecto-geniculo-cortical path, found in intact animals, ceases, which is apparently due to transsynaptic changes in the surface layers of the optic tectum.